Metabolic versatility of aerobic methane‐oxidizing bacteria under anoxia in aquatic ecosystems

Abstract The potential positive feedback between global aquatic deoxygenation and methane (CH4) emission emphasizes the importance of understanding CH4 cycling under O2‐limited conditions. Increasing observations for aerobic CH4‐oxidizing bacteria (MOB) under anoxia have updated the prevailing paradigm that MOB are O2‐dependent; thus, clarification on the metabolic mechanisms of MOB under anoxia is critical and timely. Here, we mapped the global distribution of MOB under anoxic aquatic zones and summarized four underlying metabolic strategies for MOB under anoxia: (a) forming a consortium with oxygenic microorganisms; (b) self‐generation/storage of O2 by MOB; (c) forming a consortium with non‐oxygenic heterotrophic bacteria that use other electron acceptors; and (d) utilizing alternative electron acceptors other than O2. Finally, we proposed directions for future research. This study calls for improved understanding of MOB under anoxia, and underscores the importance of this overlooked CH4 sink amidst global aquatic deoxygenation.


INTRODUCTION
Dissolved oxygen (DO) in aquatic ecosystems has declined over the past half-century, primarily because of global warming (Breitburg et al., 2018;Jane et al., 2021;Schmidtko et al., 2017;Zhi et al., 2023).Besides reducing the solubility of O 2 , greenhouse gasdriven warming raises metabolic rates and results in accelerating aquatic O 2 consumption (Keeling et al., 2010).Meanwhile, warming-induced intensified stratification accounts for significant O 2 loss by impeding ventilation, hindering O 2 transport from the surface to deeper layers (Helm et al., 2011).Moreover, once exposed to hypoxic or anoxic conditions due to deoxygenation, methanogens potentially activate the production of CH 4 , a greenhouse gas which is 28 times more potent in holding heat than carbon dioxide (CO 2 ) on a centennial timescale (Tollefson, 2022).This means that deoxygenation is likely to exert positive feedback on CH 4 emission (Bonaglia et al., 2022;Chronopoulou et al., 2017).Given that aquatic ecosystems contribute nearly half of global CH 4 emission (Rosentreter et al., 2021), even slight deoxygenation may trigger serious ecological consequences.
CH 4 emission is a balance between production and oxidation (He et al., 2018;Zhu et al., 2020).Depending on whether the electron acceptor is O 2 , CH 4 oxidation can be divided into aerobic and anaerobic CH 4 oxidation.Since O 2 is a thermodynamically favourable electron acceptor for CH 4 oxidation, aerobic CH 4 -oxidizing bacteria (MOB), a group of bacteria that grow on CH 4 as their sole source of carbon and energy (Kalyuzhnaya et al., 2019), are considered a critical biofilter to mitigate CH 4 emission (Mao et al., 2022).In the absence of O 2 , anaerobic CH 4 -oxidizing archaea (ANME) consume CH 4 via a reverse methanogenic pathway coupled with other electron acceptors like sulfate (SO 4 2À ), nitrate (NO 3 À ), and metal oxides (Boetius et al., 2000;Raghoebarsing et al., 2006;Beal et al., 2009;Haroon et al., 2013), and the NC10-bacteria related to Candidatus Methylomirabilis oxyfera (M.oxyfera) produce O 2 intracellularly from nitrite (NO 2 À ) for CH 4 oxidation (Ettwig et al., 2010).The established norm in microbial ecology is that CH 4 metabolisms by MOB are O 2 -dependent; thus, MOB are traditionally believed to thrive only in oxic environments, especially at the oxic-anoxic interface where diffusion of CH 4 from below and DO from above provide a suitable niche for them (Reim et al., 2012).Nevertheless, increasing studies have demonstrated that MOB can survive and even actively metabolize CH 4 in environments with very low or even undetectable O 2 concentrations (Figure S1).These unexpected findings have substantially updated the understanding that the ecological amplitude of MOB is broader than previously recognized, and that the role of MOB in mitigating global warming under anoxia may be neglected (Reis et al., 2024).However, our understanding of the metabolic strategies employed by MOB under anoxia remains limited.
To fill the knowledge gap, we mapped the presence of MOB in global anoxic environments, summarized four metabolic strategies for MOB survival under anoxia, and proposed directions for future research.

UBIQUITOUS PRESENCE OF MOB IN ANOXIC ENVIRONMENTS
An absolute anoxic environment (i.e., zero O 2 concentration) cannot be directly detected because no assay has detection limit low enough, typically reaching only nanomolar level (Berg et al., 2022).However, many bacteria can sense and utilize O 2 in nanomolar concentrations that escape most detection attempts, making a vague boundary between aerobes and anaerobes (Bristow et al., 2016;Kalvelage et al., 2015;Stolper et al., 2010;Trojan et al., 2021).This capability among traditionally considered aerobes indicates that they play a broader environmental role and possess more versatile metabolic pathways than those currently recognized (Berg et al., 2022;Trojan et al., 2021).Here, we operationally define the "apparent anoxic" conditions as those where DO levels drop below the detection limit of O 2 sensing technologies (Canfield & Kraft, 2022).Recent studies have identified MOB in anoxic environments globally, especially in aquatic ecosystems including oceans, hydrothermal vents, lakes, and reservoirs (Figure 1 and Table 1).Some cultures and strains enriched or isolated from these habitats also have shown that MOB actively consume CH 4 under O 2limited or depleted conditions (Figure 1 and Table 1).Intriguingly, several studies have identified MOB as the sole CH 4 consumer with no detection of anaerobic methanotrophs (e.g., ANME-type archaea and NC10-bacteria) (Dershwitz et al., 2021;Milucka et al., 2015).Moreover, Gammaproteobacterial-MOB, such as Methylobacter and Methylomonas, are frequently found in anoxic environments (He et al., 2022;Li et al., 2023;Thamdrup et al., 2019).These MOB under anoxia play an unexpected important role in mitigating CH 4 emission.For instance, MOB were identified as responsible for 40.3% of CH 4 reduction in the anoxic sediments of Lake Fuxian (Li et al., 2023), and nearly complete consumption of CH 4 in the anoxic waters of Lake Lago di Cadagno (Milucka et al., 2015).Although these types of MOB sometimes catalyse CH 4 under anoxia non-syntrophically (e.g., Kits et al., 2015), in more cases they appear to aggregate with other microorganisms (e.g., Shi et al., 2021), indicating elaborate metabolic mechanisms are involved.

METABOLIC VERSATILITY OF MOB UNDER ANOXIA
The conventional aerobic oxidation pathway of MOB has been extensively reviewed in previous studies (Hanson & Hanson, 1996;Kalyuzhnaya et al., 2019).Briefly, initiated by CH 4 monooxygenase (MMO) to split the O O bonds under the presence of O 2 , MOB activate and oxidize CH 4 to methanol (Hanson & Hanson, 1996;Murrell et al., 2000), followed by oxidation to formaldehyde by methanol dehydrogenase (MDH) with a subunit of MxaF or XoxF (Chistoserdova, 2016;McDonald & Murrell, 1997).Formaldehyde oxidation can be catalysed by several enzymes, including the tetrahydromethanopterin-(H 4 MPT-) or tetrahydrofolate linked and formaldehyde dehydrogenases (FADH, possibly also XoxF) (Kalyuzhnaya et al., 2019).Besides being oxidized via formate to CO 2 by FADH and formate dehydrogenase (FDH), another part of formaldehyde is assimilated into the biomass via the Serine pathway or the ribulose monophosphate (RuMP) pathway (Chistoserdova et al., 2005).However, MMO inhibition under anoxia suppresses the initial oxidation step in CH 4 transformation to methanol (Roslev & King, 1995).Therefore, MOB require distinct strategies to use CH 4 as a carbon and energy source under anoxia, as summarized below (Figure 2).
In the permanently stratified Lake Lago di Cadagno, where the euphotic layer extends below the oxic-anoxic interface, abundant MOB affiliated to Gammaproteobacteria were attached to photosynthetic algae and actively consume CH 4 in the anoxic water column (Milucka et al., 2015).Likewise, Methylomonas was also enriched alongside the oxygenic NC10-bacteria in a NO 2 Àdependent anaerobic CH 4 oxidation system, indicating that Methylomonas was fuelled by O 2 through NO 2 Àderived NO dismutation pathway (Chang et al., 2021).A recent study found abundant and diverse nitric oxide dismutase (NOD) genes affiliated not only with NC10-bacteria but also with diverse heterotrophic bacteria, such as Flavihumibacter, Pseudomonas and a new order (UBA11136) of Alphaproteobacteria (Elbon et al., 2024;Zhu et al., 2017).This implies that different under anoxia in typical aquatic ecosystems.The purple, brown, blue, black, green, and red filled circle represent observations from the ocean, hydrothermal vent, lake water, lake sediment, reservoir, and enrichment culture or isolated strain from the above habitats respectively.
T A B L E 1 Aerobic methane-oxidizing bacteria (MOB) under anoxia in typical aquatic ecosystems and laboratory systems.combinations between MOB and oxygenic bacteria may widely occur in anoxic environments.Additionally, in a CH 4 -based membrane biofilm batch reactor that used perchlorate (ClO 4 À ) as the electron acceptor for anaerobic CH 4 oxidation, multiple MOB genera, including Methylococcus, Methylomonas, and Methylocystis accounted for 20%-27% of the total bacteria and were likely involved in CH 4 oxidation (Lv et al., 2019).Since perchlorate-reducing bacteria reduce ClO 4 À to chlorite (ClO 2 À ), which is further intracellularly disproportionated to chloride (Cl À ) and O 2 (Miller et al., 2014), MOB could use O 2 produced by perchlorate respiration to oxidize CH 4 and instant O 2 recycling would maintain anoxic conditions.Because numerous heterotrophic bacteria have a capacity to produce ROS (Diaz et al., 2013), which can be detoxified to O 2 by superoxide dismutase (SOD) and catalase (CAT) with hydrogen peroxide (H 2 O 2 ) as the intermediate product (Apel & Hirt, 2004), it is speculated that MOB can use ROS-detoxified O 2 to oxidize CH 4 under anoxia.However, to the best of our knowledge, the detoxification of ROS coupled with CH 4 oxidation mediated by MOB has not been reported up to now.

Habitats
Self-generation/storage of O 2 by MOB (Figure 2ii) Self-generation of O 2 by MOB is dependent on the extracellular copper-binding peptides called methanobactins (MBs, Figure 3) (Dershwitz et al., 2021).Based on MBs, some Alphaproteobacterial-MOB have novel acquisition systems for copper ions (Cu 2+ ) (DiSpirito et al., 2016), that are critical for regulating MMO expression (Murrell et al., 2000).Intriguingly, MBs can bind with multiple metal ions besides Cu 2+ and reduce some bound ions (Choi et al., 2006;Lu et al., 2017).By using an H 2 18 O tracing method, two Alphaproteobacterial-MOB strains, Methylosinus trichosporium OB3b and Methylocystis sp.strain SB2, were found to produce 36 O 2 when incubated in the presence of either Au 3+ , Cu 2+ , or Ag + (Dershwitz et al., 2021).Furthermore, 36 O 2 was generated by coupling Fe 3+ reduction and H 2 18 O oxidation with the help of MBs (Dershwitz et al., 2021).This is the first evidence verifying "self-generation" of O 2 by MOB and the ability to express MBs (thereby generate O 2 ) may be an important pathway for facilitating CH 4 removal under anoxia.Notably, most MOB found in anoxic zones are affiliated to Gammaproteobacteria (Figure 1 and Table 1).Although some Gammaproteobacterial-MOB can secrete copper-binding compounds (Choi et al., 2010), none of these have been verified to have genes encoding MBs biosynthesis as of yet (Semrau et al., 2020).Therefore, it is possible that Gammaproteobacterial-MOB generate O 2 via some unknown mechanism or utilize O 2 produced by others through MBs production.Similarly, AOA, which have long been considered O 2 -dependent, were also recently found  2004).
to self-produce O 2 (Kraft et al., 2022).These studies indicate that "self O 2 generation" maybe an overlooked capacity for conventional aerobic microorganisms surviving anoxic environments.Storage of O 2 by MOB mainly relies on the O 2 -carrier bacteriohemerythrin (Figure 3), which shuttles O 2 from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by particulate MMO (Chen et al., 2012).Under O 2 -limited conditions, one notable change in the transcriptomes of MOB, such as Methylomonas denitrificans FJG1, Methylococcus capsulatus (Bath), and Methylomicrobium buryatense 5GB1C, is the upregulation of genes encoding bacteriohemerythrin (Chen et al., 2012;Gilman et al., 2017;Kits et al., 2015).This means that O 2 from episodically inputs like turbidity currents from surface water into the anoxic zone, is likely stored or consumed quickly instead of being detected during sampling campaigns (Blees et al., 2014).However, further field evidences are required to reveal the importance of O 2 storage by MOB for CH 4 removal under anoxic conditions.
Forming a consortium with non-oxygenic heterotrophic bacteria that use other electron acceptors (Figure 2iii) MOB can transform CH 4 to low molecular weight compounds via the pyrophosphate-mediated glycolytic pathway under micro-oxic conditions (Kalyuzhnaya et al., 2013;Khanongnuch et al., 2023).These compounds can be utilized as carbon sources by heterotrophic bacteria, establishing a syntrophic link between methanotrophy and heterotrophy (He et al., 2015).Moreover, trace O 2 seems to only trigger the reaction instead of acting as an electron acceptor for CH 4 oxidation, because the amount of O 2 consumption is much lower than that of CH 4 oxidation.In a membrane biofilm reactor, MOB transformed CH 4 to methanol and excreted part of it out of the cells, which was further assimilated by methanol-utilizing denitrifiers at a low O 2 :CH 4 ratio (0.06, Xu et al., 2020).
Syntrophy is not limited to micro-oxic environments.Even under anoxic conditions, MOB can still form a consortium with heterotrophic bacteria.Some field investigations found that MOB may couple CH 4 oxidation with NO x À reduction in the anoxic water columns of freshwater lakes (Rissanen et al., 2018;van Grinsven et al., 2021).An in situ observation also revealed the co-existence of MOB and iron reducers below the sulfate-methane transition zone (SMTZ) in the sediment of a boreal estuary, indicating a potential linkage between them by using Fe(III) as an alternative electron acceptors (Myllykangas et al., 2020).When solid electron acceptors such as Fe(III) oxides dominate anoxic environments, extracellular electron transfer (EET), including putative multiheme c-type cytochromes (MHCs), electrically conductive pili (e-pili), and electron shuttles (such as flavins, phenazine, and rebredoxin), is likely to play a critical role (Shi et al., 2016).Our recent study showed that the pilA gene encoding e-pili, the genes encoding putative and extracellular periplasmic MHCs, and the genes encoding electron shuttles particularly riboflavin, including ribA, ribBA, ribD, ribE, ribF, and ribH, were all present in Methylomonas (Figure 3, Li et al., 2023).Furthermore, MOB form a consortium with some heterotrophic bacteria to utilize ferrihydrite as an alternative electron acceptor under anoxia with the help of riboflavin (Li et al., 2023).Given the ubiquitous presence of electron shuttles and low molecular weight compounds in natural anoxic sediments, it is possible that similar MOB consortia exist and function in situ.This has been further verified in Arctic lake sediments, where MOB actively oxidize 13 CH 4 and generate intermediates like methanol, formaldehyde, and formate, which fuel ferric reduction via dissimilatory iron-reducing bacteria (He et al., 2022).In an anoxic membrane biofilm batch reactor, MOB excrete some fermentation by-products including formate, acetate, propionate, butyrate, and lactate for heterotrophic bacteria (such as Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae), and the latter reduce selenate to selenite and elemental selenium by proteins annotated as periplasmic NO 3 À reductases (Shi et al., 2021).
Utilizing alternative electron acceptors other than O 2 (Figure 2iv) Previous studies have shown that anaerobic methanotrophs can link CH 4 oxidation to the reduction of alternative electron acceptors under anoxia (Cai et al., 2021;Oni & Friedrich, 2017).In addition to the conventional O 2 -dependent pathway for CH 4 oxidation, some MOB have the potential to use alternative electron acceptors such as NO x À and Fe(III) under anoxia when oxygenic organisms are absent (Kits et al., 2015;Zheng et al., 2020).It has been reported that a Gammaproteobacterial-MOB Methylomonas denitrificans sp.nov.strain FJG1 T , couples CH 4 oxidation to NO 3 À reduction when DO was undetectable, releasing N 2 O as a terminal product (Kits et al., 2015).Transcriptomic analysis further revealed the upregulation of genes encoding the denitrification pathway upon NO 3 À amendment under anoxia within this MOB strain (Kits et al., 2015).Some acidophilic Alphaproteobacterial-MOB strains, such as Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6, possess N 2 O reductase genes and were recently shown to consume CH 4 under anoxia using N 2 O as the terminal electron acceptor (Awala et al., 2024).However, the genetic potential for N 2 O respiration by Gammaproteobacterial-MOB, which are ubiquitously distributed in anoxic aquatic systems, remains constrained.Besides coupling CH 4 oxidation and NO 3 À /N 2 O reduction, key genes encoding N 2 fixation (nifDHK) were present within the genome of MOB in freshwater lakes and oxygen minimum zones of the oceans (Figure 3, Jayakumar & Ward, 2020;Rissanen et al., 2021;Khanongnuch et al., 2022), indicating that MOB also have genetic potential for nitrogen fixation under O 2 -limited environments (Murrell & Dalton, 1983).Additionally, MOB strains belonging to Alpha-and Gammaproteobacteria, Methylosinus sp.LW4 and Methylomonas sp.LW13, were found to couple ferrihydrite reduction with CH 4 oxidation under O 2 limitation (initial DO of 0.89 mg/L).Although genes encoding outer membrane cytochromes were absent within Methylomonas sp.LW13, the expression of one conspicuous gene cluster encoding the Type 1 secretion system (T1SSs) was upregulated (Figure 3, Zheng et al., 2020), which is characterized by the transport of proteins from the cytoplasm to the outside of the cell and is potentially involved in EET (Kanonenberg et al., 2013;Thomas et al., 2014).Recently, a repertoire of genes encoding sulfur oxidation (Figure 3, sox-YZAB, dsrABEFHCMKLJOPN, sqr, sorAB, tetH, and doxAD) within the genome of Methylovirgula thiovorans strain HY1 suggested potential utilization of various reduced sulfur compounds for growth (Gwak et al., 2022).However, no genetic or experimental evidence of SO 4 2À reduction has been found in MOB until now, perhaps because of the low thermodynamic energy yield to support life from this reaction under anoxia (Table S1, Knittel & Boetius, 2009).

FUTURE PERSPECTIVES
Firstly, new branches of MOB have been found continuously over the past two decades (Schmitz et al., 2021), indicating that the full complement of methanotroph diversity is not yet known (Ahmadi & Lackner, 2024).Observations of MOB in anoxic aquatic ecosystems have updated the paradigm that MOB can only inhabit oxic conditions (Reis et al., 2024).Thus, it is necessary to expand investigations in global anoxic areas to unveil new members, especially the oxygen minimum zone of oceans and anoxic layers of deep lakes.Secondly, the source of the oxygen atom remains enigmatic when CH 4 is transformed to CO 2 using alternative electron acceptors by MOB independently or in combination with other microorganisms (Shi et al., 2021) (Figure 2iii,iv).Given the ancient atmosphere was characterized by limited O 2 but abundant CH 4 (Kasting, 1993), metabolic flexibility under anoxia besides conventional aerobic pathway maybe an important lifestyle for MOB before O 2 was present on Earth.Therefore, clarifying the source of the oxygen atom is helpful in revealing the mechanisms of adaptation to hypoxic and anoxic environments and in understanding the metabolic strategies of MOB in ancient atmospheric circumstances.Additionally, with more MOB strains isolated from anoxic aquatic environments, the corresponding genomic information is needed to construct their synergistic evolutionary history with Earth, especially during the Great Oxygenation Event (GOE) (Lyons et al., 2014).Thirdly, although recent case studies have shown that CH 4 oxidation mediated by MOB under anoxia significantly reduces CH 4 emission in freshwater lakes (Li et al., 2023;Milucka et al., 2015), it is urgent to re-evaluate the contribution of MOB to CH 4 mitigation on a larger anoxic scale in aquatic ecosystems, which account for half of the global CH 4 emission (Rosentreter et al., 2021).Lastly, if NO X À are the terminal electron acceptors, Gammaproteobacterial-MOB may lead to a net production of the far more potent greenhouse gas N 2 O (Griffis et al., 2017;Stein & Lidstrom, 2024), resulting in further climate change even though CH 4 is consumed (Kits et al., 2015).Therefore, it is necessary to consider the net greenhouse effect of CH 4 oxidation by MOB under O 2 -limited conditions.Given the positive feedback between greenhouse CH 4 emission and ubiquitous aquatic deoxygenation (Bonaglia et al., 2022), the role of MOB in anoxic environments needs to be thoroughly understood.
/S): depth for MOB present in the anoxic zone of water column (W) or sediment (S), with unit of m and cm, respectively.b Metabolic pathways: (a) forming a consortium with oxygenic microorganisms; (b) self-generation/ storage of O 2 by MOB; (c) forming a consortium with non-oxygenic heterotrophic bacteria that use other electron acceptors; (d) utilizing alternative electron acceptors other than O 2 .c Accession number is only for (meta)genomic sequencing data but not for amplicon sequencing data.d O 2 detection limit from Labasque et al. (

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I G U R E 2 Conventional aerobic metabolic pathway in oxic environments and four potential metabolic strategies under anoxia for MOB: (i) forming a consortium with oxygenic organisms; (ii) selfgeneration/storage of O 2 by MOB; (iii) forming a consortium with nonoxygenic heterotrophic bacteria that use other electron acceptors; (iv) utilizing alternative electron acceptors other than O 2 , such as NO X À , N 2 O, and Fe(III).